Camk2a-tTA;TRE-hShh mice overexpress dually-lipidated hShh-Np and enhance Shh signaling in forebrain. A Scheme of generating Camk2a-tTA;TRE-hShh mice. The active Camk2a promoter induces tTA expression that drives TRE-hShh to express Zsgreen1 and hShh. B Western blot of recombinant hShh-Np, 6xHis tagged mShh-N, lysate of MEF co-transfected with CMV-rtTA and the targeting vectors plus Dox treatment, and cerebral cortex lysate of TRE-hShh and Camk2a-hShh mice. The anti-hShh (C9C5) and anti-b-actin antibodies were used. C MALDI-TOF MS of recombinant protein. 6xHis tagged mShh-N (top) and hShh-Np (bottom). D Representative fluorescence microscopy images of TRE-hShh and Camk2a-hShh brains of 2-month-old mice. Both bright-field (BF) and GFP channels were shown. E Representative tile scan confocal images of a sagittal brain section of a 2-month-old Camk2a-hShh mouse. GFP channel was shown. F Zsgreen1-positive cells in Camk2a-hShh hippocampus. See Additional files 1 and 2: Video S1 and S2 for details. G FACS of dissociated cells from cerebral cortex and hippocampus of 2-month-old TRE-hShh and Camk2a-hShh mice. The same gate setting was used to separate Zsgreen1-negative and Zsgreen1-positive cells for all samples, and n = 2 per group. H Taqman RT-PCR of Shh signaling pathway genes (hShh, mShh, mGli1, and mPTCH1). Cerebral cortex, hippocampus, and cerebellum were compared. The b-actin was used for the RT-PCR normalization. Data were represented as mean ± SEM and analyzed by two-way ANOVA and Sidak’s multiple comparisons tests, and n = 5 per group. See also Additional files 1 (Video S1), 2 (Video S2), 11 (Figure S2).